1. Technical Field
The invention concerns the field of cell culture technology, specifically production host cell lines containing vector constructs comprising a ceramide transfer protein (CERT) expression cassette. Those cell lines have improved secretion characteristics in comparison to non-transgenic cell lines.
2. Background
The market for biopharmaceuticals for use in human therapy continues to grow at a high rate with over 900 biopharmaceuticals being evaluated in clinical studies and estimated sales of 50 billions in 2010. Over the years, an increasing number of biopharmaceuticals is produced from mammalian cells due to their ability to correctly process and modify human is proteins. Successful and high yield production of biopharmaceuticals from mammalian cells is thus crucial and depends on the characteristics of the recombinant monoclonal cell line used in the process.
Since most biopharmaceutical products are proteins that are secreted from the cells during the production process, the secretory transport machinery of the production cell line is another interesting target for novel host cell engineering strategies.
Protein secretion is a complex multi-step mechanism: Proteins destined to be transported to the extracellular space or the outer plasma membrane are first co-translationally imported into the endoplasmic reticulum. From there, they are packed in lipid vesicles and transported to the Golgi apparatus and finally from the trans-Golgi network (TGN) to the plasma membrane where they are released into the culture medium.
Many engineering approaches have employed the growing understanding of the molecular networks that drive processes such as transcription and translation to increase the yield of these steps in protein production. However, as for any multi-step production process, widening a bottle-neck during early steps of the process chain possibly creates bottle necks further downstream, especially post translation. Up to a certain threshold, the specific productivity of a production cell has been reported to correlate linearly with the level of product gene transcription. Further enhancement of product expression at the mRNA level, however, may lead to an overload of the protein synthesis, folding or transport machinery, resulting in intracellular accumulation of the protein product. Indeed, this can be frequently observed in current manufacturing processes.
Therefore, there is a need for improving the secretory capacity of host cells for recombinant protein production. This might even become more important in combination with novel transcription-enhancing technologies and in high-titer processes in order to prevent post-translational bottle necks and intracellular accumulation of the protein product.
However, previous approaches to target the post-translational machinery, have not succeeded but rather led to contradictory results depending on cell line or product used in the study or the initial productivity level:                Overexpression of the ER-resident molecular chaperone BiP (binding protein BiP/GRP78) unexpectedly resulted in reduced secretion;        Enhanced expression of the enzyme protein disulfide isomerase (PDI) showed contradictory results.        
Secretion engineering using the transcription factor X-box binding protein 1 (XBP-1) was observed to either have no effect or to enhance secretion, however, apoptotic cell death was increased concomitantly, leading to an instable phenotype and preventing the isolation of XBP-1 high-expressing clones.
Thus, at present, there are two major hurdles on the way to targeted manipulation of the secretory transport machinery: The still limited knowledge about the underlying regulatory mechanisms and the requirement to prevent a concomitant growth-inhibitory or apoptotic response of the producer cell.